Structure-activity relationship (SAR) studies on substituted N-(pyridin-3-yl)-2-amino-isonicotinamides as highly potent and selective glycogen synthase kinase-3 (GSK-3) inhibitors.

In our continuing efforts to explore structure-activity relationships around the novel class of potent, isonicotinamide-based GSK3 inhibitors described in our previous report, we extensively explored structural variations around both 4/5-pyridine substitutions and the amide group. Some analogs were found to have greatly improved pTau lowering potency while retaining high kinase selectivity. In contrast to previous active compounds 1a-c, a close analog 3h did not show in vivo efficacy in a triple-transgenic mouse Alzheimer's disease model. In general, these 2­pyridinyl amide derivatives were prone to amidase mediated hydrolysis in mouse plasma.

Enhanced IL-36R signaling promotes barrier impairment and inflammation in skin and intestine.

Deficiency in interleukin-36R (IL-36R) antagonist caused by loss-of-function mutations in IL-36RN leads to DITRA (deficiency of IL-36 receptor antagonist), a rare inflammatory human disease that belongs to a subgroup of generalized pustular psoriasis (GPP). We report a functional genetic mouse model of DITRA with enhanced IL-36R signaling analogous to that observed in patients with DITRA, which provides new insight into our understanding of the IL-36 family of molecules in regulating barrier integrity across multiple tissues. Humanized DITRA-like mice displayed increased skin inflammation in a preclinical model of psoriasis, and in vivo blockade of IL-36R pathway using anti-human IL-36R antibody ameliorated imiquimod-induced skin pathology as both prophylactic and therapeutic treatments. Deeper characterization of the humanized DITRA-like mice revealed that deregulated IL-36R signaling promoted tissue pathology during intestinal injury and led to impairment in mucosal restoration in the repair phase of chronic dextran sulfate sodium (DSS)-induced colitis. Blockade of IL-36R pathway significantly ameliorated DSS-induced intestinal inflammation and rescued the inability of DITRA-like mice to recover from mucosal damage in vivo. Our results indicate a central role for IL-36 in regulating proinflammatory responses in the skin and epithelial barrier function in the intestine, suggesting a new therapeutic potential for targeting the IL-36R axis in psoriasis and at the later stages of intestinal pathology in inflammatory bowel disease.

Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors.

GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics.

Dual role for Fcγ receptors in host defense and disease in Borrelia burgdorferi-infected mice.

Arthritis in mice infected with the Lyme disease spirochete, Borrelia burgdorferi, results from the influx of innate immune cells responding to the pathogen in the joint and is influenced in part by mouse genetics. Production of inflammatory cytokines by innate immune cells in vitro is largely mediated by Toll-like receptor (TLR) interaction with Borrelia lipoproteins, yet surprisingly mice deficient in TLR2 or the TLR signaling molecule MyD88 still develop arthritis comparable to that seen in wild type mice after B. burgdorferi infection. These findings suggest that other, MyD88-independent inflammatory pathways can contribute to arthritis expression. Clearance of B. burgdorferi is dependent on the production of specific antibody and phagocytosis of the organism. As Fc receptors (FcγR) are important for IgG-mediated clearance of immune complexes and opsonized particles by phagocytes, we examined the role that FcγR play in host defense and disease in B. burgdorferi-infected mice. B. burgdorferi-infected mice deficient in the Fc receptor common gamma chain (FcεRγ(-/-) mice) harbored ~10 fold more spirochetes than similarly infected wild type mice, and this was associated with a transient increase in arthritis severity. While the elevated pathogen burdens seen in B. burgdorferi-infected MyD88(-/-) mice were not affected by concomitant deficiency in FcγR, arthritis was reduced in FcεRγ(-/-) MyD88(-/-) mice in comparison to wild type or single knockout mice. Gene expression analysis from infected joints demonstrated that absence of both MyD88 and FcγR lowers mRNA levels of proteins involved in inflammation, including Cxcl1 (KC), Xcr1 (Gpr5), IL-1beta, and C reactive protein. Taken together, our results demonstrate a role for FcγR-mediated immunity in limiting pathogen burden and arthritis in mice during the acute phase of B. burgdorferi infection, and further suggest that this pathway contributes to the arthritis that develops in B. burgdorferi-infected MyD88(-/-) mice.

The caspase 1 inflammasome is not required for control of murine Lyme borreliosis.

The contribution of the inflammasome to the development of immune responses and disease during infection with the Lyme disease spirochete, Borrelia burgdorferi, is not well defined. Host defense against the spirochete is severely impaired in mice deficient in the adaptor molecule myeloid differentiation antigen 88 (MyD88), which is required not only for Toll-like receptor-mediated responses but also for the production of the proforms of interleukin 1beta (IL-1beta) and IL-18. These cytokines are released in active forms after cleavage by the inflammasome-associated enzyme caspase 1. To investigate the contribution of the inflammasome to host defense against B. burgdorferi, we examined Lyme borreliosis in mice deficient in either caspase 1 or apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), a molecule upstream of caspase 1 in the inflammasome signaling cascade. We found that caspase 1-deficient mice had a mild transient elevation in pathogen burden and a trend toward an increase in the prevalence of arthritis early in infection, but these differences resolved by day 14 postinfection. Caspase 1 deficiency had no effect on B. burgdorferi-induced humoral immunity, T-cell responses, or the abilities of macrophages to ingest and degrade spirochetes. The absence of the ASC protein had no effect on the control of the spirochete or the development of immune responses and disease. These findings reveal that the caspase 1 inflammasome is not critical to host defense against the extracellular pathogen Borrelia burgdorferi.

MyD88 deficiency enhances acquisition and transmission of Borrelia burgdorferi by Ixodes scapularis ticks.

Borrelia burgdorferi strains exhibit various degrees of infectivity and pathogenicity in mammals, which may be due to their relative ability to evade initial host immunity. Innate immune cells recognize B. burgdorferi by Toll-like receptors (TLRs) that use the intracellular molecule MyD88 to mediate effector functions. To determine whether impaired TLR signaling enhances Ixodes scapularis acquisition of B. burgdorferi, we fed nymphs on wild-type (WT) and MyD88-/- mice previously infected with two clinical isolates of B. burgdorferi, BL206, a high-virulence strain, and B348, an attenuated strain. Seventy-three percent of the nymphs that fed on BL206-infected WT mice and 40% of the nymphs that fed on B348-infected WT mice acquired B. burgdorferi, whereas 100% of the nymphs that fed on MyD88-/- mice became infected, irrespective of B. burgdorferi strain. Ticks that acquired infection after feeding on MyD88-/- mice harbored more spirochetes than those that fed on WT mice, as assessed by quantitative PCR for B. burgdorferi DNA. Vector transmission of BL206 and B348 was also enhanced when MyD88-/- mice were the blood meal hosts, with the mean pathogen burden at the skin inoculation site significantly higher than levels in WT mice. These results show that the absence of MyD88 facilitates passage of both low- and high-infectivity B. burgdorferi strains between the tick vector and the mammal and enhances the infectivity of a low-infectivity B. burgdorferi strain.

Myeloid differentiation antigen 88 deficiency impairs pathogen clearance but does not alter inflammation in Borrelia burgdorferi-infected mice.

The spirochete Borrelia burgdorferi causes acute inflammation in mice that resolves with the development of pathogen-specific adaptive immunity. B. burgdorferi lipoproteins activate innate immune cells via Toll-like receptor 2 (TLR2), but TLR2-deficient mice are not resistant to B. burgdorferi-induced disease, suggesting the involvement of other TLRs or non-TLR mechanisms in the induction of acute inflammation. For this study, we used mice that were deficient in the intracellular adapter molecule myeloid differentiation antigen 88 (MyD88), which is required for all TLR-induced inflammatory responses, to determine whether the interruption of this pathway would alter B. burgdorferi-induced disease. Infected MyD88(-/-) mice developed carditis and arthritis, similar to the disease in wild-type (WT) mice analyzed at its peak (days 14 and 28) and during regression (day 45). MyD88(-/-) macrophages produced tumor necrosis factor alpha only when spirochetes were opsonized, suggesting a role for B. burgdorferi-specific antibody in disease expression. MyD88(-/-) mice produced stronger pathogen-specific Th2-dependent immunoglobulin G1 (IgG1) responses than did WT mice, and their IgM titers remained significantly elevated through 90 days of infection. Despite specific antibodies, the pathogen burden was 250-fold higher in MyD88(-/-) mice than in WT mice 45 days after infection; by 90 days of infection, the pathogen burden had diminished substantially in MyD88(-/-) mice, but it was still elevated compared to that in WT mice. The elevated pathogen burden may be explained in part by the finding that MyD88(-/-) peritoneal macrophages could ingest spirochetes but degraded them more slowly than WT macrophages. Our results show that MyD88-dependent signaling pathways are not required for B. burgdorferi-induced inflammation but are necessary for the efficient control of the pathogen burden by phagocytes.